瓯柑中性转化酶基因(CrInv1)克隆及序列特征分析

Cloning and Sequence Analysis of a Neutral Invertase Gene(CrInv1) from Ougan Fruit

研究旨在克隆瓯柑中性转化酶基因(Inv),了解其序列特征,为从分子生物学水平研究瓯柑果实蔗糖代谢提供参考。采用同源克隆和RT-PCR技术对瓯柑Inv基因进行扩增,得到的基因命名为CrInv1,基因登录号为KF694988。用生物信息学方法对获得的序列进行预测蛋白结构域、系统进化分析以及分子量、等电点分析,获得瓯柑转化酶基因序列信息。克隆获得CrInv1基因全长为1932bp的cDNA序列,预测编码643个氨基酸。序列比对结果显示,在不同物种中Inv基因高度保守,CrInv1与NCBI网站上其他物种的序列同源性均高于70%。Primer5.0软件预测CrInv1蛋白的相对分子量为72.18kDa,理论等电点(pI)为6.882,为中性转化酶蛋白,富含非极性氨基酸,亚细胞定位在膜内。系统进化树结果显示,瓯柑CrInv1蛋白与草莓、荔枝等的亲缘关系较为接近,而与葡萄、桃、苹果的Inv蛋白分属不同分支。试验获得了瓯柑CrInv1基因及相关序列信息,为后续瓯柑果实蔗糖代谢分子生物学研究提供了参考。

The objective of this study was to separate Inv gene from ougan fruit and get sequence information,which would be used in further study on sucrose metabolism of ougan fruit at molecular level.Methods ofhomologous cloning and RT-PCR were used forInv gene clone from ougan fruit.The gene we got was named CrInv1 and the accession number was KF694988.Bioinformatics methods were used to get information ofpredicted Inv protein including relative molecular mass,isoelectric point（p I）,the conserved domains andphytogenetic analysis results.Results showed that CrInv1 was 1932 bp in size,encoding a putative protein of643-amino acid.Sequence alignment results showed that CrInv1 was highly conserved among different plants,and shared more than 70% sequence homology with other plants from NCBI.With the calculation by softwarePrimer 5.0,relative molecular mass of the putative CrInv1 protein turned out to be 72.18 k Da,and the p I was6.882.Results indicated that CrInv1 from ougan fruit belonged to neutral invertase and it was rich in nonpolaramino acids.The subcellular localization of predicted CrInv1 protein was within the cell membrane.Phylogenetic analysis showed that putative CrInv1 protein was not grouped with that from grape,apple andpeach,and was more close to that from lichi and strawberry.The study got Inv gene from ougan fruit and thesequence information was analyzed.The results lay a foundation for subsequent research on sucrosemetabolism of ougan fruit.